Chinese Journal of Physiology 58(5): 312-321, 2015
DOI: 10.4077/CJP.2015.BAD317
Role of Efferent Sympathoadrenal Effects in
Cooling-Induced Hemodynamic Perturbations
in Rats: An Investigation by Spectrum Analysis
Yia-Ping Liu 1, Yi-Hsien Lin 3, 4, Chen-Cheng Lin 1, Yu-Chieh Lin 1, Yu-Chun Chen 1,
Po-Lei Lee 2, and Che-Se Tung 4
1
Department of Physiology, National Defense Medical Center, Taipei 11490
Department of Electrical Engineering, National Central University, Taoyuan 32001
3
School of Medicine, National Yang-Ming University, Taipei 11221
and
4
Division of Medical Research and Education, Cheng Hsin General Hospital, Taipei 11280
Taiwan, Republic of China
2
Abstract
Cold stress may produce hemodynamic perturbations but the underlying mechanisms are still
not clear. Spectral analysis was used in this study to explore that sympathoadrenal activation could
be involved in mechanisms of hemodynamic perturbations to cooling. Conscious rats after treatment
with a control vehicle (saline) compared with withdrawal of sympathetic influences by ganglion blocker
hexamethonium (HEX) or chemical sympathectomy guanethidine (GUA) were challenged by stressful
cooling as acute immersing all four extremities in ice water (4 ± 2°C) for 10 min. Plasma nitric oxide (NO)
and the appearance of Dichroitic notch (DN) were measured in comparison between treatment groups
throughout the experimental course. Hemodynamic indices were telemetrically monitored, and variability of blood pressure and heart rate (BPV; HRV) were assessed over a range of frequencies: very-low
frequency (VLF: 0.02-0.2 Hz), low frequency (LF: 0.2-0.6 Hz), high frequency (HF: 0.6-3 Hz), normalized (n)LF, nHF, ratio LF/HF of HRV (LF/HFHRV), and total power (TP: ≦3 Hz). Results showed that
the concomitant reciprocal changes of spectral powers existed between frequencies of BPV and HRV to
the stressful cooling (i.e. VLFBPV versus VLFHRV, LFBPV versus LFHRV, and nLFBPV versus nLFHRV) which
contribute to the underlying mechanisms of sympathetic efferent influences and myogenic cardiovascular responsiveness. Furthermore, compared with the control vehicle in the stressful cooling, HEX
restrained the increase of the pressor, tachycardia and VLF BPV, except that VLF HRV was reduced. GUA
abolished pressor, however, restrained the increase of the tachycardia, VLF BPV and LF BPV. In addition,
GUA reversed the downward tendency of nLF BPV into an upward tendency and attenuated both
nLF HRV and LF/HF HRV. DN was virtually undetectable after HEX management but was apparently
noticeable after GUA management. Finally, the increase of plasma NO after cooling was diminished
after HEX or GUA management. Taken together, these results substantiate that the spectral changes
during stressful cooling are highly relevant to the efferent sympathetic rhythmicity and subsequent
NO production.
Key Words: guanethidine, hemodynamic perturbations, hexamethonium, spectral powers, stressful
cooling, sympathetic influences
Corresponding author: Che-Se Tung, M.D., Ph.D., Division of Medical Research and Education, Cheng Hsin General Hospital, No. 45, Cheng
Hsin St, Beitou, Taipei 11280, Taiwan, R.O.C. Tel: +886-2-28264400 ext. 7037, E-mail: [email protected]
Received: August 25, 2014; Revised: November 3, 2014; Accepted: December 15, 2014.
2015 by The Chinese Physiological Society and Airiti Press Inc. ISSN : 0304-4920. http://www.cps.org.tw
312
Sympathoadrenal Efferent and Stressful Cooling
Introduction
Stressful cooling elicits an immediate increase
in arterial blood pressure (BP), heart rate (HR) and
greater adrenergic neurotransmissions, which is commonly used in clinical practice in diagnosis on autonomic control of the cardiovascular system in cold
pressor test (18); however, there is little substantiated evidence for the underlying mechanisms of this
test.
It is well known that when immediately exposed
to cooling, a predominant initial response is restricted
blood flow due to cooling-induced vasoconstriction.
As exposure to cooling persists, the peripheral blood
flow passes through a phase of induced vasodilation
thought to be a protective mechanism against tissue
damage (4, 12, 14).
The interplay between initial vasoconstriction
and the subsequently evoked vasodilatation through
prolonged cooling is complex (4, 6), which requires
intact sympathetic and sensory functions as well as
ensuing compensatory baroreflex and releases of
humoral substances. The evoked vasodilatation, a
myogenic vascular responsiveness, is suggested by
reduction of vascular resistance and increases of nitric oxide (NO) production (8, 11, 21, 23).
Spectral analysis in hemodynamic fluctuations
has been widely applied to investigate the baroreflex
control of cardiovascular homeostasis—a dynamic
interplay between ongoing BP perturbation and compensatory cardiovascular response (2, 5, 10, 16). The
standard frequencies commonly used for power assessment in BP variability (BPV) and HR variability
(HRV) are the following: (a) the high frequency (HF)
BPV (HFBPV) reflects the oscillation in cardiac output
secondary to mechanic respiratory sinus arrhythmia,
whereas HF of HRV (HF HRV) reflects oscillation in
respiration and efferent vagal modulation of HR; (b)
the low frequency (LF) of BPV (LFBPV) reflects oscillation in efferent sympathetic modulation of vascular
resistance, whereas LF of HRV (LF HRV) reflects oscillation in sympathetic modulation of HR; (c) the LF/HF
ratio of HRV (LF/HF HRV) reflects the overall balance
between efferent sympathetic and vagal modulations
of HR; (d) the total power (TP) of BPV (TPBPV) or HRV
(TPHRV) reflects the variance of oscillations and overall
autonomic activity in vascular bed or the heart; and
(e) the very-low frequency (VLF) of BPV (VLFBPV) or
HRV (VLF HRV), a heterogeneously frequency band,
the physiological background of which is mostly unrevealed. However, this band has been variously ascribed to the thermoregulatory vasomotor modulation,
activity of hormonal systems and the autonomic nervous
system itself (7, 13, 17, 20).
Since the regulation of sympathetic outflow can
be highly patterned in baroreflex mechanisms of hemo-
313
dynamic changes, the question arises as to how different
patterns of sympathetic activation are generated by
various kinds of pathophysiological stimuli (3, 9).
We have previously successfully developed a rodent
model to study cooling stress through a novel measurement of cardiovascular variability, and the results
revealed that frequency power of VLFBPV might reflect
the myogenic vascular responses to the cooling-induced
sympathetic activation (22). In addition, the coolinginduced spectral VLFBPV enhancement has been related
to sympathetic activation and subsequent NO production. The present study was performed through
pharmacological intervention to explore the underlying mechanisms of cooling-induced spectral power
VLFBPV enhancement in which the efferent sympathoadrenal pathways could be involved.
Materials and Methods
Subjects
Adult male Sprague-Dawley rats (BioLASCO,
Taiwan, ROC) weighing between 300 and 350 g were
used. The experiments were performed according to
a protocol approved by the Animal Care Committee
of the National Defense Medical Center, Taipei. All
efforts were made to keep the number of animals
used as low as possible and to minimize animal suffering during the experiments. All rats were housed
in a temperature- and humidity-controlled holding
facility with a 12-hour light/dark cycle, with light
on from 07:00 to 19:00, maintained by manual light
control switches as required by the experiment. Rats
in the same experimental group were housed together.
All rats received food and water ad libitum. Experiments were performed between 08:30 and 17:30, with
all rats were tested at the same time every day, whenever possible.
Experimental Protocols and Cooling Procedure
Rats were randomly divided into three groups in
assessing the effects of withdrawal of the neural
sympathetic influences: (a) in the control vehicle intervention by intraperitoneal (i.p.) injection of saline
(n = 12) to obtain hemodynamic data in the intact
condition; (b) after induction of ganglionic blockade
by hexamethonium (HEX) (30 mg/kg intravenous
bolus followed by continuous infusion at 1.5 mg/
kg/min) (n = 12), and the infusion was continued
throughout the experimental course; (c) after sympathetic denervation by i.p. injection of guanethidine
sulfate (GUA) (50 mg/kg i.p. seven times a week for
1 week) (n = 12), and the treatment was continued
throughout the experimental course. The stressful
cooling stimulation consisted of rapidly immersing
314
Liu, Lin, Lin, Lin, Chen, Lee and Tung
all four extremities of an individual rat in ice-cold
water (CI).
Prior to the experiments, the rats were adapted to
the experimental circumstances for approximately 30
min. Following a complete stabilization of hemodynamics and temperature, an individual rat was quickly
placed in a Plexiglas cage with ice-water (depth, 2
cm; temperature, 4°C) and received CI for a period
of 10 min. After exposure, the rat was removed from
the cage and dried with a cloth in a similar cage for
30 min to facilitate recovery. Hemodynamic changes
were monitored continuously via a telemetric device
in 10-min stages, i.e., 10 min before (PreCI), 20 min
after (PostCI), and during the 10-min of the cooling
trial (0-3 min: CIIP, the initial ascending pressor
period; 7-10 min: CIMP, the maintaining plateau
pressor period). Dicrotic notch (DN) was counted
manually.
Surgical Intervention
A telemetry transmitter was implanted intraabdominally into each rat under anesthesia (sodium
pentobarbital, 50 mg/kg). A laparotomy was performed
under aseptic procedure and the catheter of the transmitter was inserted into the abdominal aorta, distal to
the kidneys, and fixed. Experiments began after the
rat had fully recovered from the surgery in 7 days.
Acquisition and Processing of Spectrum Signals
On the day of the experiment, the pulse signals
obtained after magnetic activation of the transmitter
at least 1 h before starting the test were generated
as a calibrated analog signal (UA10; DSI, St. Paul,
MN, USA) with a range of ± 5 V and a 12-bit resolution. Individual rat in each group was then placed
on the top of a receiver (PhysioTel ® RPC-1) for acquisition of telemetric signals. Five receivers were
connected to a PC desktop computer via a matrix
(Dataquest ART Data Exchange Matrix) and the signals were recorded with the Dataquest Acquisition
software (Dataquest ART 4.33). A series of successive signals of SBP and inter-beat interval (IBI) throughout the experiments were then digitized at a 500-Hz
sampling rate and processed off-line using Matlab
software (Terasoft Co. San Diego, CA).
Beat-by-beat fluctuations in SBP and IBI movements were analyzed to quantitate the frequency and
power in cardiovascular variability (BPV and HRV)
using autoregressive spectral decomposition. Spectral
analysis for BPV calculation was based on a software
kindly written for us by Professor P.L. Lee, National
Central University, Taiwan. Briefly, the acquired SBP
signals were pre-processed by applying a band-pass
filter (0.1~18 Hz, zero-phase 4th-order) to remove DC
components. After finding all the maximum SBP peaks
between two zero-cross points, the extracted beat-bybeat SBP time series were detrended, interpolated and
resampled at 0.05 s to generate a new time series of
evenly spaced SBP sampling, which allowed a direct
spectral analysis of each distribution using a Fast
Fourier Transform (FFT) algorithm. On the other hand,
spectral analysis for HRV calculation was based on the
Chart software developed by PowerLab, ADInstruments, Colorado Springs, CO, USA. Spectral indexes
of BPV and HRV were then computed independently
to obtain the total power (0.00 to 3.0 Hz, TP) and
three major spectral components: very-low frequency
(0.02 to 0.2 Hz, VLF), low frequency (0.20 to 0.60 Hz,
LF) and high frequency (0.60 to 3.0 Hz, HF). The
normalized LF and HF were also calculated as nLF
or nHF = LF or HF/TP-VLF × 100%. The modulus of
the HR or BP spectrum (ordinates) had units of ms 2
or mmHg 2.
NO Assay
Tail venous blood was withdrawn for NO assessments in the following rats: Vehicle-PreCI (n = 6):
rats treated with control vehicle and blood was withdrawn before CI (PreCI); Vehicle-CI (n = 6): rats treated
with control vehicle and blood was withdrawn immediately after 10 min of a CI trial; HEX-CI (n = 6):
rats treated with HEX and blood was withdrawn immediately after a 10-min CI trial. GUA-CI (n = 6): rats
treated with GUA and blood was withdrawn immediately after a 10-min CI trial. The plasma NO level was
determined indirectly by the content of nitrite and
nitrate using enzyme-linked immunosorbent assay
(ELISA) kits (CAT No. 780001) supplied by Cayman
Chemical Company (Ann Arbor, MI, USA). Results
were expressed as μM.
Statistical Analysis
Statistical analyses were performed using the
SPSS version 18.0 software. Homogeneity of variance was assessed by the Kolmogorov-Smirnov test.
Statistical comparisons were performed by repeated
measures two-way ANOVA followed by a post-hoc
Scheffe’s test (SPSS version 18.0). Student’s t-test was
used to detect differences between two groups. A Pvalue < 0.05 was considered statistically significant.
The results are expressed as mean ± SEM.
Results
Typical examples of BP tracings and the changes
in power spectrum are shown in Figs. 1 and 2 for rats
receiving pharmacological interventions. Averaged
data are shown in Figs. 3-5.
315
Sympathoadrenal Efferent and Stressful Cooling
PreCI
CIIP
CIMP
PostCI
Control
200 mmHg
60 mmHg
0 sec
60 sec
0 sec
60 sec 0 sec
60 sec
0 sec
60 sec
0 sec
60 sec
0 sec
60 sec 0 sec
60 sec
0 sec
60 sec
0 sec
60 sec
0 sec
60 sec 0 sec
60 sec
0 sec
60 sec
HEX
200 mmHg
60 mmHg
GUA
200 mmHg
60 mmHg
Fig. 1. A typical example of blood pressure tracings in rats treated with saline (Control Vehicle) or withdrawal of sympathetic influences
by HEX or GUA before the cooling stimulation. Abbreviations: Stressful cooling as rapid immersion of four extremities in
4°C ice water (CI), 10 min before CI (PreCI), 20 min after CI (PostCI), and during 10 min of CI (0-3 min: CIIP, the initial
ascending pressor period; 7-10 min: CIMP, the maintaining plateau pressor period).
Cooling-Induced SBP and HR with Concomitant DN
Changes
The changes in cooling-induced SBP and HR
are shown in Fig. 3. After ganglionic blockade with
the HEX treatment, cooling generally induced pressor (PreCI versus CIIP, P < 0.001) and tachycardia
(PreCI versus CIIP and CIMP, all P < 0.001). However, after sympathetic denervation with the GUA treatment, cooling induced tachycardia at CIIP (PreCI
and PostCI versus CIIP, all P < 0.001) but without
SBP changes comparing with PreCI and PostCI.
Compared with the control vehicle, HEX generally
significantly increased HR but attenuated SBP in
both the PreCI and PostCI stages (P < 0.01~0.001),
but conversely, GUA generally caused no significantly changes but tended to attenuate both HR and
SBP in both the PreCI and PostCI stages. However,
during CI exposure, all HEX and GUA generally significantly attenuated SBP and HR at both CIIP and
CIMP (P < 0.001).
The appearance of DN showed that when compared to the control vehicle during CI exposure, HEX
caused DN virtually undetectable at both CIIP (0.03 ±
0.03% versus 3.94 ± 1.48%) and CIMP (0.0 ± 0.0%
versus 0.64 ± 0.41%), but conversely, GUA generally
increased the appearance of DN (CIIP: 55.73 ± 14.95%
versus 3.94 ± 1.48%; CIMP: 33.63 ± 15.38% versus
0.64 ± 0.41%).
Cooling-Induced Spectral Power Changes
The global changes of spectral powers of VLF,
LF and nLF in BPV and HRV are selectively shown in
Fig. 4. During CI exposure, HEX and/or GUA tended
to increase VLF BPV (HEX: PreCI and PostCI versus
CIIP, all P < 0.05, PreCI versus CIMP, P = 0.112; GUA:
PreCI versus CIIP, P = 0.219, PreCI versus CIMP, P =
0.188) (Fig. 4A), LFBPV (HEX: PreCI and PostCI versus
CIIP and CIMP, all P < 0.05; GUA: PreCI and PostCI
versus CIIP, P < 0.05) (Fig. 4B), nLFBPV (GUA: PreCI
and PostCI versus CIIP and CIMP, P < 0.05~0.01)
(Fig. 4C) and nLF HRV (HEX: PostCI versus CIMP,
P < 0.05) (Fig. 4C), and TP BPV (HEX: PreCI–7.59 ±
3.15 and PostCI–8.09 ± 2.0 versus CIIP–28.63 ± 9.94,
all P < 0.01; GUA: PreCI–3.66 ± 0.81 vs. CIIP–31.22 ±
15.7, P < 0.05), whereas both HEX and GUA tended
to decrease VLF HRV (HEX: PostCI versus CIIP and
CIMP, all P < 0.05; GUA: PreCI and PostCI versus
CIIP, P = 0.615~0.688) (Fig. 4A).
Compared to the control vehicle prior to CI initiation (PreCI), HEX significantly attenuated VLF HRV
(Fig. 4A; P < 0.01), nLF BPV and nLFHRV (Fig. 4C; all
P < 0.05), LF/HFHRV (0.04 ± 0.01 versus 0.19 ± 0.03,
P < 0.05), and TP HRV (20.35 ± 8.37 versus 40.25 ±
7.44, P < 0.05), whereas GUA significantly attenuated
VLFBPV (Fig. 4A; P < 0.05), LFBPV (Fig. 4B; P < 0.01),
nLFBPV (Fig. 4C; P < 0.01), nLFHRV (Fig. 4C; P < 0.05),
and TPBPV (3.66 ± 0.81 versus 6.76 ± 1.68, P < 0.05).
In contrast, when compared with the control
vehicle during the CI exposure, HEX significantly
attenuated VLF BPV and VLF HRV (CIIP: all P < 0.05
and CIMP: all P < 0.01) (Fig. 4A), LF BPV (CIIP: P <
316
Liu, Lin, Lin, Lin, Chen, Lee and Tung
Control Vehicle
HRV
VLF LF
HF
BPV
VLF LF
HF
PreCI
PreCI
CIIP
CIIP
ms2
mmHg2
CIMP
CIMP
PostCI
PostCI
0.02 0.2 0.6
3
0.02 0.2 0.6
Frequency (Hz)
3
Frequency (Hz)
HEX
HRV
VLF LF
HF
GUA
BPV
VLF LF
HF
HRV
VLF LF
HF
BPV
VLF LF
HF
PreCI
PreCI
CIIP
CIIP
ms2
ms2
mmHg2
mmHg2
CIMP
CIMP
PostCI
PostCI
0.02 0.2 0.6
3
Frequency (Hz)
0.02 0.2 0.6
3
Frequency (Hz)
0.02 0.2 0.6
3
Frequency (Hz)
0.02 0.2 0.6
3
Frequency (Hz)
Fig. 2. A typical example of spectral power changes in rats treated with saline (Control Vehicle) or withdrawal of sympathetic influences by HEX or GUA before the cooling stimulation. Blood pressure variability (BPV) and heart rate variability (HRV)
were assessed over a range of frequencies: very-low frequency (VLF: 0.02-0.2 Hz), low-frequency (LF: 0.2-0.6 Hz), highfrequency (HF: 0.6-3 Hz). Abbreviations used are as described in the legend to Fig. 1.
0.01) (Fig. 4B), LF/HFHRV (CIMP: 0.08 ± 0.02 versus
0.23 ± 0.07, P < 0.01), and TPBPV (CIIP: 28.63 ± 9.94
versus 146.82 ± 46.3, P < 0.05 and CIMP: 19.63 ± 20.19
versus 88.51 ± 35.18, P < 0.05), whereas GUA significantly increased TP HRV (CIIP: 49.8 ± 8.33 versus
21.15 ± 4.43, P < 0.05 and CIMP: 45.64 ± 9.18 versus
25.33 ± 6.56, P < 0.05), attenuated VLFBPV (CIIP: P <
0.05 and CIMP: P < 0.05) (Fig. 4A), LFBPV (CIIP: P <
0.01 and CIMP: P < 0.05) (Fig. 4B), nLFHRV (CIMP:
P < 0.01) (Fig. 4C), LF/HF HRV (CIMP: 0.10 ± 0.04
versus 0.23 ± 0.07, P < 0.05), and TP BPV (CIIP:
31.22 ± 15.70 versus 146.82 ± 46.30, P < 0.05).
NO Levels
After 10 min of a CI trial, the significant elevation of plasma NO in the rats treated with the control
vehicle was diminished compared with the vehiclePreCI and also in the rats treated with either HEX
or GUA (Fig. 5; P < 0.05).
Discussion
As expected, the pattern of hemodynamic changes
under control conditions in the present study was con-
317
Sympathoadrenal Efferent and Stressful Cooling
Control Vehicle
HEX
GUA
HR
**
**
**
600
500
**
¶
‡ ¶
‡
ll
‡
160
140
**
#
**
200
¶
‡
‡
120
300
**
**
¶
‡
**
‡
100
80
60
40
100
0
**
**
180
mmHg
beats/min
400
¶
‡ ¶
‡
**
**
**
SBP
20
PreCI
CIIP
CIMP
PostCI
0
PreCI
CIIP
CIMP
PostCI
Fig. 3. The effects of stressful cooling on systolic blood pressure (SBP) and heart rate (HR) in rats treated with saline (Control Vehicle)
(n = 12) or withdrawal of sympathetic influences by HEX (n = 12) or GUA (n = 12) before the cooling stimulation. Significant differences between PreCI and CI stages (‡P < 0.001) or between PostCI and CI stages (||P < 0.01, ¶P < 0.001) were
evaluated by repeated measures two-way ANOVA and post-hoc Scheffe’s test. Significant differences in rats between control
vehicle and HEX or control vehicle and GUA (**P < 0.01~0.001) were evaluated by Student’s t-test. Abbreviations used are
as described in the legend to Fig. 1.
sistent to our previous findings (22). Stressful acute
cooling significantly perturbed the hemodynamic pattern of the tested rats, as evidenced by pressor, tachycardia and concomitant reciprocal changes in spectral
powers between frequencies of BPV and HRV, which
contribute to the underlying mechanisms in the activation of sympathetic efferent influences and myogenic cardiovascular responsiveness (13, 17). Owing
to the fact that the rhythmic oscillations of the LF band
is referred as a marker for the status of sympathetic
modulation of cardiovascular tonicity, whereas the
VLF band is referred as an indicator of cardiovascular myogenic responsiveness to hemodynamic perturbations (13, 17, 19, 20), we will focus on such two
bands in the following discussion.
The Effects of Neural Sympathetic Influences
In sympathetic modulation, the changes were examined after withdrawal of the neural sympathetic
influences by HEX or GUA. Under treatment with
the control vehicle, results showed that during CI exposure, the absolute value, “LF BPV”, markedly increased, whereas the relative unit, “nLF”, both of
nLF HRV and nLF BPV, were reduced. When compared
with the control vehicle, results showed that both HEX
and GUA significantly attenuated both SBP and HR
throughout the CI trial. However, HEX remained the
pressor and tachycardia to the cooling stimulation,
whereas GUA abolished the pressor, attenuated but
remained the tachycardia to the cooling stimulation.
Both HEX and GUA significantly attenuated but
remained the increasing tendency of LF BPV to the
cooling stimulation, and HEX and GUA both also
attenuated nLF HRV and LF/HF HRV during the later
cooling period (CIMP). On the other hand, when compared with the control vehicle throughout the experiment course, HEX did not cause much effective
nLF BPV changes; however, GUA reversed the downward tendency of nLF BPV into an upward tendency.
In general, these results are in keeping with the notion that sympathetic vasoconstrictor system can be
involved in the cooling-induced pressor responses,
and additional aspects of these results deserve further comments.
Firstly, it is to be noted that our discussion is
based on the presumption that neural sympathetic
influences on vasculature and the heart were completely eliminated in resting condition (PreCI) after
HEX or GUA treatment. However, we have unexpectedly observed pressor and/or tachycardia, which
still remained even after removal of the neural influences. During the cooling stimulation of the rats
treated with ganglionic blockade (HEX), both pressor
and tachycardia largely diminished but still existed.
On the other hand, during the cooling stimulation of
the rats treated with sympathetic denervation (GUA),
both pressor and tachycardia were clearly reduced,
and the pressor was totally abolished; however, tachycardia was reduced but still existed.
For these observed changes after HEX treatment,
two possible mechanisms may be involved: (a) some
318
Liu, Lin, Lin, Lin, Chen, Lee and Tung
HRV
25
PSD (ms2)
20
**
**
**
15
**
10
0
**
**
*
*
PreCI
*
15
#
**
§
*
10
§
§
§
CIIP
CIMP
0
PostCI
PreCI
2.0
15
#
1.5
1.0
PSD (mmHg2)
PSD (ms2)
25
#
¶
‡
0.0
PreCI
CIIP
CIMP
C. nLF
0.10
#
#
#
0.30
0.08
§
n.u.
0.06
#
PostCI
#
§
§
*
CIIP
CIMP
* *
**
**
PreCI
*
#
PostCI
#
**
**
**
#
#
§
*
0.20
n.u.
§
*
0.04
0.25
#
*
10
0
PostCI
CIMP
ll
‡
15
5
0.5
CIIP
**
**
B. LF
2.5
#
*
*
*
5
§
5
#
#
§
25
PSD (mmHg2)
A. VLF
BPV
Control Vehicle
HEX
GUA
‡
§
†
0.15
0.10
0.02
0.00
0.05
PreCI
CIIP
CIMP
PostCI
0.00
PreCI
CIIP
CIMP
PostCI
Fig. 4. The power spectral density (PSD) changes in (A) very-low frequency bands (VLF) and (B) low-frequency bands (LF), and (C)
The relative unit (n.u.) changes in normalized low-frequency band (nLF) of blood pressure variability (BPV) or heart rate
variability (HRV) of rats treated with saline (Control Vehicle) (n = 12) or withdrawal of sympathetic influences by HEX (n =
12) or GUA (n = 12) before the cooling experiments. Significant differences between PreCI and CI stages (*P < 0.05) or
between PostCI and CI stages (¶P < 0.001) were evaluated by repeated measures two-way ANOVA and post-hoc Scheffe’s
test. Significant differences in rats between control vehicle and HEX or control vehicle and GUA (#P < 0.05, **P < 0.01~0.001)
were evaluated by Student’s t-test.
unknown effects originated from the central nervous
system which might have elicited and augmented
the efferent sympathetic rhythmicity to vascular beds
and the heart, and such central effects consequently
overdrawn the resting blockade effects of HEX; (b)
local cooling might have activated the cold-sensitive
afferents to some downstream somatosympathetic relaying areas, such as the dorsal root ganglion, where
they are not blocked after HEX treatment.
For the observed changes after GUA treatment,
Sympathoadrenal Efferent and Stressful Cooling
4
an upward tendency is due to an electrical/chemical dissociation phenomenon, i.e., the efferent sympathetic
fibers are still intact, action potential still appears,
but the efferent sympathetic fibers lose the baroreflex
function to compensate the acute stressful cooling, a
phenomenon analogous to an engine idling.
2
The Effects of Cardiovascular Myogenic Responsiveness
0
In cardiovascular myogenic responsiveness to
the cooling stimulation, power change of VLF bends
between BPV and HRV was studied after withdrawal
of sympathetic influences by HEX or GUA. There was
an immediate increase of VLF BPV but a decrease of
VLF HRV during treatment with the control vehicle;
however, when HEX or GUA was applied compared
with the control vehicle treatment, the VLFBPV bands
were generally attenuated but remained an increasing
tendency to the cooling stimulation. On the other
hand, when compared with control vehicle to the cooling stimulation, HEX attenuated VLF HRV throughout
the experimental course , but GUA generally did not
change much, on the contrary, by a rising propensity
in CIIP.
An association between VLF BPV band and vascular myogenic responsiveness has been reported previously (13, 17, 19, 20). The reports suggested that
because sympathetic activation and blood flow fluctuations could lead to dynamic BP fluctuations as increase of VLF BPV spectral power, and this increase
of VLFBPV spectral power could be due to the increase
of vascular myogenic responsiveness. In addition, it
is well known that the ongoing blood flow and BP
perturbations could produce mechanical shear stress
and enhance endothelial releases of NO (19), this
secondary humoral substance could then antagonize
the initial rising of BP to protect endothelial cells
from hemodynamic injuries (8, 15).
Consequently, as for the aforementioned attenuation of VLFBPV after either HEX or GUA treatment,
we believe that the attenuation is a consequence of
the deterioration of the cooling-evoked sympathetic
activation on resistance vessels. As for the still existing
rising propensity of VLF BPV to the cooling stimulation, our explanation is that even if the neural sympathetic influences on vascular beds were completely
eliminated, there might still be some unknown humoral substances present to enhance the rhythmic
oscillation in vasculature.
Nevertheless, our data further showed that, as
compared with control vehicle treatment, during the
cooling stimulation, both sympathetic withdrawal treatments have concurrently attenuated the spectral powers
of LFBPV and VLFBPV; in addition, after HEX or GUA
treatment, the effects on NO production were abolished.
Since the aforementioned sympathetic activation could
10
Nitrate + Nitrite (uM)
319
8
*
Vehicle - PreCI
Vehicle - CI
HEX - CI
GUA - CI
6
Fig. 5. Plasma nitric oxide (NO) levels of rats treated with
saline (Control Vehicle) (n = 6) or withdrawal of sympathetic influences by HEX (n = 6) or GUA (n = 6) before the cooling experiments. Significant differences
between treatments were evaluated by Student’s t-test
(*P < 0.05). See legend to Fig. 1 for abbreviations
used.
a possible explanation is based on the pharmacological features of this drug, i.e. once GUA has entered
the sympathetic terminals for sympathectomy, it blocks
the release of norepinephrine in response to arrival
of an action potential; however, spontaneous release
is not affected. In addition, GUA is known to have
spare effects on central noradrenergic neurons and
adrenal medulla (1). Therefore, these two interesting
hemodynamic changes observed during the cooling
stimulation after GUA treatment can be explained as
follows: (a) the pressor had totally abolished and tachycardia still existed but had been reduced, and (b) the
downward tendency of nLFBPV of control vehicle was
reversed into an upward trend. The former possibility
may be explained by the adrenal medulla-spared function of GUA that the remained tachycardia is possible
because of the cooling-evoked releases of epinephrine
from the adrenal medulla into the circulation. The
latter possibility is further discussed in more details
below:
The normalized LF (nLF) was used in this study
as an index to illustrate the efferent sympathetic influences of either sympathetic vascular outflow
(nLF BPV) or sympathetic cardiac outflow (nLF HRV),
because this application accords with the principles
of baroreflex mechanisms, i.e., the firing rhythmicity
is reasonable to be coherence between efferent sympathetic fibers to the targeted resistance vessels and
to the heart (5, 10, 16). In other words, our using
nLF BPV rather than using LF BPV is more appropriate
to signify the compensatory baroreflex effects of sympathetic influences on vascular resistance. In addition, to denote the sympathetic influences on vascular
bed, nLFBPV is reported to be specific without the influence of HFBPV (24). As a consequence, we attribute
that during the cooling stimulation after GUA treatment, the downward tendency of nLFBPV reversed into
320
Liu, Lin, Lin, Lin, Chen, Lee and Tung
enhance VLF BPV through the shear stress-dependent
NO production, our findings are in line with the previous assumption that the spectral power changes in
VLFBPV band during stressful cooling are highly relevant to the sympathetic activation and subsequent NO
production (13, 17, 19, 20).
Finally, we observed an attenuation of VLF HRV
band to the cooling stimulation only after HEX treatment, whereas VLF HRV band was not much changed
but slightly increased at CIIP after GUA treatment.
To the effects of ganglionic blocker HEX effects, our
explanation is because both sympathetic and parasympathetic influences on the cardiac muscles were
eliminated. However, to the effect of chemical sympathectomy GUA effects, we speculate here that it was
because the adrenal medulla-spared function again, the
expedited circulatory epinephrine and β-adrenoceptor
activation might have enhanced the rhythmic oscillation in the myocardiac functions. Furthermore, our
data proved that DN was virtually undetectable after
ganglionic blocker HEX treatment, but DN was apparently noticeable after chemical sympathectomy
GUA treatment. Taken together, our findings support that autonomic balance and humoral substance
were both involved in the cardiac effects as for the
spectral changes of VLF HRV band during the cooling
stimulation.
Conclusions
The use of power spectrum analysis has enabled
applications in assessing stressful cooling-induced
hemodynamic perturbations. The present study has
demonstrated that the spectral changes during cooling
stimulation are highly relevant to the efferent sympathetic rhythmicity and subsequent NO production.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Acknowledgments
We thank Dr André Diedrich for helpful comments
during preparation of the article, and Miss Chan-Fan
Young for establishing the animal surgery for telemetry and spectrum analysis and in performing the experiments. This study was funded by grants from the
National Science Council (NSC 102-2320-B-016-014)
and the National Defense Medical Center (MAB10140; MAB-102-81), Taipei, Taiwan, ROC.
17.
18.
19.
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